Submicron streptavidin patterns for protein assembly

Micron and submicron-scale features of aldehyde functionality were fabricated in polymer films by photolithography to develop a platform for protein immobilization and assembly at a biologically relevant scale. Films containing the pH-reactive polymer poly(3,3'-diethoxypropyl methacrylate) and a photoacid generator (PAG) were patterned from 500 nm to 40 mum by exposure to 365 nm (i-line) light. Upon PAG activation and hydrolysis of acetals, aldehyde groups formed. After the films were incubated with a biotinylated aldehyde reactive probe, the X-ray photoelectron spectroscopy results were consistent with biotin being attached to the surface. The background was subsequently passivated by flood exposure and incubation with an aminooxy-terminated poly(ethylene glycol), resulting in a 98% reduction in nonspecific protein adsorption. Protein patterning and assembly was demonstrated using streptavidin, biotinylated anthrax toxin receptor-1, and the protective antigen moiety of anthrax toxin and confirmed by fluorescence microscopy and atomic force microscopy (AFM). AFM demonstrated that 500 nm protein features were achieved. Because of the abundance of biotinylated proteins, this methodology provides a platform for protein immobilization and assembly for various applications in biotechnology.     

Fully automated molecular mechanics based induced fit protein-ligand docking method

We describe a method for docking a ligand into a protein receptor while allowing flexibility of the protein binding site. The method employs a multistep procedure that begins with the generation of protein and ligand conformations. An initial placement of the ligand is then performed by computing binding site hotspots. This initial placement is followed by a protein side-chain refinement stage that models protein flexibility. The final step of the process is an energy minimization of the ligand pose in the presence of the rigid receptor. Thus the algorithm models flexibility of the protein at two stages, before and after ligand placement. We validated this method by performing docking and cross docking studies of eight protein systems for which crystal structures were available for at least two bound ligands. The resulting rmsd values of the 21 docked protein-ligand complexes showed values of 2 A or less for all but one of the systems examined. The method has two critical benefits for high throughput virtual screening studies. First, no user intervention is required in the docking once the initial binding site selection has been made in the protein. Second, the initial protein conformation generation needs to be performed only once for a given binding region. Also, the method may be customized in various ways depending on the particular scenario in which dockings are being performed. Each of the individual steps of the method is fully independent making it straightforward to explore different variants of the high level workflow to further improve accuracy and performance.     

Heterotelechelic Polymers for Capture and Release of Protein-Polymer Conjugates

A heterotelechelic biotin-maleimide polymer containing a cleavable disulfide bond was synthesized by RAFT polymerization and used to reversibly modify surfaces with proteins.     

Measuring particle size-dependent physicochemical structure in airborne single walled carbon nanotube agglomerates

As-produced single-walled carbon nanotube (SWCNT) material is a complex matrix of carbon nanotubes, bundles of nanotubes (nanoropes), non-tubular carbon and metal catalyst nanoparticles. The pulmonary toxicity of material released during manufacture and handling will depend on the partitioning and arrangement of these components within airborne particles. To probe the physicochemical structure of airborne SWCNT aggregates, a new technique was developed and applied to aerosolized as-produced material. Differential Mobility Analysis-classified aggregates were analyzed using an Aerosol Particle Mass Monitor, and a structural parameter Γ (proportional to the square of particle mobility diameter, divided by APM voltage) derived. Using information on the constituent components of the SWCNT, modal values of Γ were estimated for specific particle compositions and structures, and compared against measured values. Measured modal values of Γ for 150 nm mobility diameter aggregates suggested they were primarily composed of non-tubular carbon from one batch of material, and thin nanoropes from a second batch of material – these findings were confirmed using Transmission Electron Microscopy. Measured modal values of Γ for 31 nm mobility diameter aggregates indicated that they were comprised predominantly of thin carbon nanoropes with associated nanometer-diameter metal catalyst particles; there was no indication that either catalyst particles or non-tubular carbon particles were being preferentially released into the air. These results indicate that the physicochemistry of aerosol particles released while handling as-produced SWCNT may vary significantly by particle size and production batch, and that evaluations of potential health hazards need to account for this. Disclaimer: The mention of any company or product does not constitute an endorsement by the Centers for Disease Control and Prevention. The findings and conclusions in this paper are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health.     

In situ preparation of protein-"smart" polymer conjugates with retention of bioactivity

Protein-polymer conjugates are widely used in biotechnology and medicine, and new methods to prepare the bioconjugates would be advantageous for these applications. In this report, we demonstrate that bioactive "smart" polymer conjugates can be synthesized by polymerizing from defined initiation sites on proteins, thus preparing the polymer conjugates in situ. In particular, free cysteines, Cys-34 of bovine serum albumin (BSA) and Cys-131 of T4 lysozyme V131C, were modified with initiators for atom transfer radical polymerization (ATRP) either through a reversible disulfide linkage or irreversible bond by reaction with pyridyl disulfide- and maleimide-functionalized initiators, respectively. Initiator conjugation was verified by electrospray-ionization mass spectroscopy (ESI-MS), and the location of the modification was confirmed by muLC-MSMS (tandem mass spectrometry) analysis of the trypsin-digested protein macroinitiators. Polymerization of N-isopropylacrylamide (NIPAAm) from the protein macroinitiators resulted in thermosensitive BSA-polyNIPAAm and lysozyme-polyNIPAAm in greater than 65% yield. The resultant conjugates were characterized by gel electrophoresis and size exclusion chromatography (SEC) and easily purified by preparative SEC. The identity of polymer isolated from the BSA conjugate was confirmed by (1)H NMR, and the polydispersity index was determined by gel permeation chromatography (GPC) to be as low as 1.34. Lytic activities of the lysozyme conjugates were determined by two standard assays and compared to that of the unmodified enzyme prior to polymerization; no statistical differences in bioactivity were observed.     

Instabilities of waves in nonlinear disordered media

We develop a self-consistent theory of temporal fluctuations of a speckle pattern resulting from the multiple scattering of a coherent wave in a weakly nonlinear disordered medium. The speckle pattern is shown to become unstable if the nonlinearity exceeds a threshold value. The instability is due to a feedback provided by the multiple scattering and manifests itself in spontaneous fluctuations of the scattered intensity. The development of instability is independent of the sign of nonlinearity.